CA2291883C - Trigger factor expression plasmids - Google Patents

Abstract

An artificial operon comprising genes encoding each of a trigger factor, GroEL and GroES, an expression plasmid carrying the operon, an expression plasmid carrying a gene encoding the trigger factor, a cotransformant harboring an expression vector for a foreign protein and any one of the expression plasmids, and a method for producing a foreign protein comprising expressing a foreign protein by the use of the cotransformant, which are capable of expressing a foreign protein in a solubilized form and in a state of having a correct conformation.

Description

TRIGGER FACTOR EXPRESSION PLASMIDS
BACKGROUND OF THE INVENTION
Field of the Invention The present invention relates to an artificial operon, an expression plasmid carrying the operon, a cotransformant harboring an expression vector for a foreign protein and any one of expression plasmids, and a method for producing a foreign protein comprising expressing a foreign protein by the use of the cotransformant, which are capable of expressing a foreign protein in a solubilized form and in a state of having a correct conformation.

Discussion of the Related Art A trigger factor is a protein which has been found as a cytoplasmic factor required for in vitro transporting to a membrane of proOmpA, a precursor of E. coli outer membrane protein OmpA [Crooke, E. and Wickner, W., Proc.
Nat. Acad. Sci. USA 84, 5216-5220 (1987)]. In addition, a tig gene has been cloned as a gene encoding a trigger factor having a molecular weight of 48 kDa [Guthrie, B.
and Wickner W., J. Bacteriol. 172, 5555-5562 (1990)]. On the basis of analysis of the amino acid sequence, it has been elucidated that the trigger factor has FK506-bound protein (FKBP) domain, and that all of amino acid residues required for each expression of the binding activity with FK506 and for a peptidyl-prolyl isomerase (PPIase) activity are conserved in the trigger factor [Callebaut, I. and Mornon, J.-P., FEBS Lett. 374, 211-215 (1995)].

There has been reported that the trigger factor has been also identified as PPIase bound to 50S subunit of E.
coli ribosome, and that the trigger factor markedly enhances prolyl isomerization in in vitro refolding of mutant RNase T1 [Stoller, G. et al., EMBO J. 14, 4939-4948 (1995)]. Moreover, there has been found by an experiment using a crosslinking reagent that the trigger factor is bound to a nascent polypeptide chain on E. coli ribosome [Valent, Q. A. et al., EMBO J. 14, 5494-5505 (1995);

Hesterkamp, T. et al., Proc. Nat. Acad. Sci. USA 93, 4437-4441 (1996)]. In addition, the trigger factor has been known to enhance binding to an unfolded protein of GroEL [Kandror, 0. et al., EMBO J. 14, 6021-6027 (1995);
Kandror, 0. et al., J. Biol. Chem. 272, 1730-1734 (1997)].

PPIase acts on proline residue in a peptide chain, and catalyzes cis-trans isomerization of conformation regarding a peptide bond. This reaction is considered as a rate-determining step of a folding process of the protein. In addition, it is considered that the PPIase family protein is involved in protein folding, refolding, association and dissociation, transport, and the like, within the cells.

In addition, the trigger factor is shown to assist the folding of several proteins in vitro [Scholz, C. et al., EMBO J. 16, 54-58 (1997)]. However, the actual function of the trigger factor has not yet been known.
In expression of a foreign protein by E. co1i, various efforts have been made on the aggregation suppression and the stabilization of a desired foreign protein by coexpression of chaperones. However, it has been difficult to predict coexpression of which of the chaperones is effective for a particular protein, so that undue experimentation is presently carried out in order to determine the effective chaperone. In addition, there are some cases where sufficient effects cannot be obtained by coexpression of known chaperones.

In view of the above problems, an object of the present invention is to provide an artificial operon comprising genes encoding each of a trigger factor, GroEL

and GroES, the artificial operon being capable of expressing a foreign protein in a solubilized form and in a state of having a correct conformation.

In one embodiment, the present invention provides an expression plasmid carrying the operon and an expression plasmid for a trigger factor.

In another embodiment, the present invention provides a cotransformant harboring both of the above expression plasmid and an expression vector for a foreign protein.
In still another embodiment, the present invention provides a method for producing a foreign protein comprising expressing a foreign protein by the use of the cotransformant.

Certain exemplary embodiments can provide an artificial operon comprising a gene encoding a trigger factor, a gene encoding GroEL, and a gene encoding GroES.

These and other objects of the present invention will be apparent from the following description.

SUMMARY OF THE INVENTION

In sum, the present invention pertains to the following:

[1] an artificial operon comprising genes encoding each of a trigger factor, GroEL and GroES;

[2] a plasmid capable of expressing each of a trigger factor, GroEL and GroES, the plasmid carrying the artificial operon according to item [1];

[3] a plasmid capable of expressing a trigger factor, the plasmid carrying a gene encoding the trigger factor under control of an inducible promoter;

[4] a cotransformant harboring the plasmid according to item [2] or [3] and an expression plasmid for a foreign protein; and [5] a method for producing a foreign protein comprising expressing said foreign protein by the cotransformant according to item [4].

BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will become more fully understood from the detailed description given hereinbelow and the accompanying drawings which are given by way of illustration only, and thus, are not limitative of the present invention, and wherein:

Figure 1 is a schematic view showing pTf13 (about 5.9 kb), wherein araB p/o is araB promoter/operator, tig is a structural gene for a trigger factor, pACYC ori is an origin of replication derived from pACYC plasmid, Cmr is chloramphenicol resistance gene, and araC is araC
activator/repressor, respectively;

Figure 2 is a schematic view showing pG-Tfl (about 8.4 kb), wherein Pzt-lp is Pzt-1 promoter, groES and groEL
are genes each encoding GroES and GroEL, tig is a structural gene for a trigger factor, tetR is tetR
repressor, Cmr is chloramphenicol resistance gene, and pACYC ori is an origin of replication of pACYC plasmid, respectively;

Figure 3 shows the analytical results on SDS-PAGE of solubilization of murine endostatin by coexpression with a trigger factor, wherein S is a soluble fraction, and I is an insoluble fraction; and Figure 4 shows the analytical results on SDS-PAGE of solubilization of human ORP150 by coexpression with a trigger factor, wherein S is a soluble fraction, and I is an insoluble fraction.

DETAILED DESCRIPTION OF THE INVENTION

One of the great features of the artificial operon of the present invention resides in that the operon comprising genes encoding each of a trigger factor, GroEL

and GroES (the genes respectively referred to as tig gene, groEL gene and groES gene). Since the artificial operon of the present invention comprises each of tig gene, groEL
gene and groES gene mentioned above, there can be exhibited an excellent effect that a soluble expression product can be efficiently obtained when the artificial operon is coexpressed with a gene encoding a foreign protein.

In the present invention, the term "trigger factor"
refers to a factor which has been found as a cytoplasmic factor required for in vitro transporting to a membrane of proOmpA, a precursor of E. coli external membrane protein OmpA.

The trigger factor is a factor having the amino acid sequence as shown in SEQ ID NO: 1 [Guthrie, B. and Wickner, W., J. Bacteriol. 172, 5555-5562 (1990)]. In the present invention, the trigger factor also encompasses a factor having a sequence in which a mutation of substitution, deletion, addition or insertion of one or more amino acid residues is introduced in the amino acid sequence as shown in SEQ ID NO: 1 mentioned above, as long as the factor is capable of expressing a foreign protein in a solubilized form and in a state of having a correct conformation by coexpression with a foreign protein encoding a foreign gene.

In the artificial operon of the present invention, there can be used tig gene corresponding to an amino acid sequence of the trigger factor. The tig gene includes a gene comprising the nucleotide sequence as shown in SEQ ID NO: 2 [Guthrie, B. and Wickner, W., J. Bacteriol.
172, 5555-5562 (1990)]. The gene comprising the nucleotide sequence as shown in SEQ ID NO: 2 can be obtained, for instance, from Kohara Clone No. 148 [Kohara, Y. et al., Cell 50, 495-508 (1987)].

In addition, in the present invention, the tig gene also encompasses a gene having a sequence in which a mutation of substitution, deletion, addition or insertion of one or more bases is introduced in the nucleotide sequence as shown in SEQ ID NO: 2 mentioned above, as long as it is a gene encoding a factor capable of expressing a foreign protein in a solubilized form and in a state of having a correct conformation by coexpression with a foreign gene.

Further, in the present invention, the tig gene also encompasses a gene comprising a DNA capable of hybridizing under stringent conditions to any one of DNAs selected from the group consisting of a DNA comprising the nucleotide sequence as shown in SEQ ID NO: 2 and a DNA
having a sequence in which a mutation of substitution, deletion, addition or insertion of one or more bases is introduced in the nucleotide sequence as shown in SEQ ID NO: 2 mentioned above, as long as it is a gene encoding a factor capable of expressing a foreign protein in a solubilized form and in a state of having a correct conformation by coexpression with a foreign gene.

The conditions for hybridization include conditions described, for instance, in Molecular Cloning: A
Laboratory Manual, 2nd Ed. [Sambrook, J. et al., Cold Spring Harbour Laboratory Press, New York (1989)], and the like.

The amino acid sequences for GroEL and GroES usable in the present invention are shown in SEQ ID NOs: 3 and 4, respectively [Hemmingsen, S. M. et al., Nature 333, 330-334 (1988)]. In the present invention, GroEL and GroES also encompass factors each having a sequence in which a mutation of substitution, deletion, addition or insertion of one or more amino acid residues is introduced in each of the amino acid sequences as shown in SEQ ID NOs: 3 and 4 mentioned above, as long as the factors have equivalent functions to wild-type GroEL and GroES each having the sequence as shown in SEQ ID NOs: 3 and 4 mentioned above, respectively.

The groEL gene and the groES usable in the present invention gene include genes comprising each of the nucleotide sequences as shown in SEQ ID NOs: 5 and 6, respectively [Hemmingsen, S. M. et al., Nature 333, 330-334 (1988)]. Each of the groEL gene and the groES
gene can be obtained, for instance, from pGroll plasmid [Nishihara, K. et al., App1. Environ. Microbiol. 64, 1694-1699 (1988)].

In the present invention, the groEL gene and the groES gene encompass genes each having a sequence in which a mutation of substitution, deletion, addition or insertion of one or more bases is introduced in each of the nucleotide sequences as shown in SEQ ID NOs: 5 and 6 mentioned above, as long as each of the genes encodes a factor having an equivalent function to wild-type GroEL
and GroES mentioned above.

Further, the groEL gene and the groES gene also encompass genes each comprising a DNA capable of hybridizing under stringent conditions to any one of DNAs selected from the group consisting of a DNA comprising the nucleotide sequence as shown in SEQ ID NO: 5 or 6 and a DNA having a sequence in which a mutation of substitution, deletion, addition or insertion of one or more bases is introduced in the nucleotide sequence as shown in SEQ ID NO: 5 or 6 mentioned above, as long as each of the genes encodes a factor having an equivalent function to wild-type GroEL and GroES mentioned above.

In the artificial operon of the present invention, the arrangement of the tig gene, the groEL gene and the groES gene is not particularly limited. Examples thereof include an operon arranged sequentially as groES-groEL-tig, and the like.

In the artificial operon of the present invention, the tig gene, the groEL gene and the groES gene can be localized under the control of a promoter.

The promoter for controlling the transcription of the operon existing under the control of the promoter is preferably an inducible promoter, from the viewpoint of regulating each of the expression levels of a trigger factor, GroEL and GroES. Examples of the inducible promoter include, for instance, lac, tac, trc, trp, ara, Pzt-1, PL and T7. Each of the lac, tac and trc promoters can be induced by using isopropyl-R-D-thiogalactopyranoside (IPTG); each of the trp, ara and Pzt-1 promoters can be induced with 3-indoleacrylic acid (IAA), L-arabinose and tetracycline, respectively; and the PL promoter can be induced at a high temperature (42 C).
Also, the T7 promoter, which is specifically and strongly transcribed by T7 RNA polymerase, can be used. In this case, the T7 promoter can be induced with IPTG by using an E. coli strain harboring a lysogenized k phage carrying the T7 RNA polymerase gene ligated downstream of the lac promoter is used. Among the promoters, from the viewpoint of facilitating the manipulations for induction, lac, trp, ara and Pzt-1 are preferable. The promoter is contained in a known vector, and it can be used by appropriately cutting out from the vector by using a restriction enzyme, and the like.

In addition, in the artificial operon of the present invention, a factor encoded by an artificial operon can be more stably expressed by possessing a terminator represented by, for instance, rrnBT1T2. The terminator is contained in a known vector, and it can be used by appropriately cutting out from the vector by using a restriction enzyme, and the like.

Concrete examples of the artificial operon of the present invention include, for instance, an operon comprising the nucleotide sequence as shown in SEQ ID NO: 7.

One of the great features of the plasmid of the present invention resides in that the plasmid carries a gene encoding a trigger factor, or it carries the artificial operon.

In the plasmid of the present invention, it is preferable that a factor encoded by a gene encoding a trigger factor, or factors (trigger factor, GroEL and GroES) encoded by an artificial operon can be expressed by using an inducible promoter.

In addition, when the plasmid of the present invention is introduced into a host, there may be used a desired foreign protein and a plasmid carrying a gene encoding a trigger factor, or a gene encoding the operon on the same plasmid. In addition, there may be used simultaneously a plasmid carrying a gene encoding a trigger factor or a gene encoding the operon, and a plasmid carrying a gene encoding a foreign protein (hereinafter referred to as "coexpression plasmid").
Among these plasmids, the coexpression plasmid is preferable from the viewpoints of not necessitating to prepare for each foreign protein a plasmid carrying a desired foreign protein and a gene encoding a trigger factor or a gene encoding the operon, and the stability of the plasmid in a host.

In order to optimize the expression level and the expression timing of the trigger factor or factors encoded by the artificial operon, without lowering the expression level of the foreign protein, it is advantageous that the expression of the trigger factor or the factors encoded by the artificial operon can be independently regulated from expression of a desired protein. From the above aspects, it is preferable that the inducible promoters used in expression of the trigger factor or the factors encoded by the artificial operon are different from the promoter usable in expression of a desired protein.

When a coexpression plasmid is used as the plasmid, any expression vector can be used, as long as it carries a replicon which does not exhibit incompatibility with an expression vector of a desired protein in a host used, including, for instance, E. co1i. For instance, when a vector carrying ColEl replicon, the vector including, for instance, pBR322 is used as an expression vector for a desired protein, the plasmid used in expression of the trigger factor or the factors encoded by the artificial operon includes p15A replicon existing in pACYC plasmid derivatives.

Concrete examples of the expression plasmid of the present invention include coexpression plasmids pTf13 and pG-Tfl. Schematic views of each of these coexpression plasmids are shown in Figures 1 and 2, respectively.

Each of pTf13 and pG-Tfl can be obtained by, for instance, the procedures set forth in Examples 1 and 2 below.

The cotransformant of the present invention can be obtained by introducing the plasmid of the present invention (coexpression plasmid) and an expression vector for a foreign protein into an appropriate host.

The expression vector for a foreign protein used in the cotransformant described above is not particularly limited. The vector includes a vector being capable of expressing in the cytosol of cells of a desired foreign protein or secreting in the periplasm of cells, and exhibiting compatibility with the coexpression plasmid.
In particular, those vectors in which expression of a desired foreign protein is induced by an inducible promoter are preferable. The inducible promoter includes the same promoters as those listed above. The trigger factor or the factors encoded by an artificial operon can be induced to be expressed independently from a desired foreign protein by selecting a promoter other than the promoter used in the induction of expression of the trigger factor or the factors encoded by an artificial operon.

In addition, the expression vector for a foreign protein may carry a selection marker gene as occasion demands. Examples of such selection marker genes include ampicillin resistance (Amp') genes, kanamycin resistance (Kmr) genes, and chloramphenicol resistance (Cmr) genes. A
double selection of the cotransformant can be made possible by using a selection marker gene different from that contained in the plasmid (coexpression plasmid) of the present invention.

The expression vector for a foreign protein is preferably a vector capable of secreting to the periplasm of cells, from the viewpoint of correct formation of disulfide bonds in the resulting foreign protein.

Examples of the expression vector include, for instance, a vector carrying a gene encoding a polypeptide resulting from addition of a signal peptide, such as OmpA, OmpT, MalE or R-lactamase, to a desired foreign protein. The expression vector can be obtained, for instance, by adding a gene encoding the signal peptide to a site corresponding to N-terminus of a desired foreign protein by genetic engineering techniques, and incorporating the resulting gene into a known vector.

In addition, in the expression vector of a foreign protein of the present invention, there may be contained a sequence which can facilitate purification of a desired protein, as represented by, for instance, expression as a fusion protein with a protein such as R-galactosidase, glutathione-S-transferase or a maltose-bound protein;

expression as histidine-tagged proteins, or the like.
The host usable in the present invention includes, for instance, E. coli strains. Concrete examples of the strains include generally employed strains such as HB101, JM109, MC4100, MG1655 and W3110; and various mutants, including protease mutants such as degP mutants, ompT
mutants, tsp mutants, 1on mutants, cIpPX mutants, hslV/U
mutants, lon-c1pPX double mutants and lon-clpPX-hslV/U
triple mutants; pisX mutants; rpoH mutants such as rpoH

deletion mutants and rpoH missense mutants, and the like.
In the present invention, protease mutants, such as lon-c1pPX double mutants and lon-clpPX-hs1V/U triple mutants; p1sX mutants; and rpoH mutants, can be favorably used, from the viewpoint of more stably expressing the foreign protein. Among the rpoH mutants, rpoH deletion mutants are preferable, from the viewpoint of more stably expressing the foreign protein.

Here, a preferable lon-c1pPX double mutant is KY2783 strain derived from E. coli strain W3110, the KY2783 resulting from introduction of double deletion mutations in the lon and c1pPX genes. The KY2783 strain was named and identified as E. coli KY2783 and has been deposited under accession number FERM BP-6244 with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, of which the address is 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan; date of original deposit: February 3, 1998.

Also, the term "Ion-clpPX-hs.IV/U triple mutant"
refers to a mutant prepared by further mutating hs1V/U
gene encoding HslV/U protease, in the above-described Ion-cIpPX double mutant. A preference is given to KY2893 strain derived from E. coli strain W3110, the KY2893 strain resulting from introduction of triple deletion mutations in the lon, c1pPX and hslV/U genes. The KY2893 strain was named and deposited E. coli KY2893 strain and has been deposited under accession number FERM BP-6243 with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, of which the address is 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan; date of original deposit: February 3, 1998.

The foreign protein to be expressed in the present invention may be any protein, as long as it is a foreign protein capable of being instabilized and/or insolubilized in E. co1i. Concrete examples of the foreign proteins include interferons, interleukins, interleukin receptors, interleukin receptor antagonists, granulocyte colony-stimulating factors, granulocyte macrophage colony-stimulating factors, macrophage colony-stimulating factors, erythropoietin, thrombopoietin, leukemia inhibitory factors, stem cell growth factors, tumor necrosis factors, growth hormones, proinsulin, insulin-like growth factors, fibroblast growth factors, platelet-derived growth factors, transforming growth factors, hepatocyte growth factors, osteogenic factors, nerve growth factors, ciliary neurotrophic factors, brain-derived neurotrophic factors, glial-derived neurotrophic factors, neurotrophine, angiogenesis inhibitors, prourokinase, tissue plasminogen activators, blood coagulation factors, protein C, glucocerebrosidase, superoxide dismutase, renin, lysozyme, P450, prochymosin, trypsin inhibitors, elastase inhibitors, lipocortin, reptin, immunoglobulins, single-chain antibodies, complement components, serum albumin, cedar pollen allergens, hypoxia-induced stress proteins, protein kinases, proto-oncogene products, transcription regulation factors and virus-constituent proteins.

The method for introducing the plasmid of the present invention into E. coli together with an expression vector for a foreign protein includes a usual method, such as calcium chloride method, rubidium chloride method or electroporation method. The cotransformant can be screened by using a reagent depending on the selection marker gene. Expression of the foreign protein can be confirmed, for instance, by Western blott analysis.

One of the great features of the method for producing a foreign protein resides in that the method comprises expressing the foreign protein by the use of the cotransformant described above. The foreign protein can be produced by, for instance, a process comprising culturing a transformant under induction conditions in which the expression level of the trigger factor or the expression level of each of trigger factor, GroEL and GroES is each at a level appropriate for stabilization and/or solubilization of a foreign protein to be expressed; harvesting the cells; disrupting the harvested cells; isolating and purifying the foreign protein from the disrupted cell solution in accordance with the purification method depending upon the desired foreign protein.

The induction conditions differ depending upon the inducible promoters used for the plasmid of the present invention and the expression vector for a foreign protein, as long as the conditions are such that the expression level of the trigger factor or the expression level of each of trigger factor, GroEL and GroES is each at a level appropriate for stabilization and/or solubilization of the foreign protein. For instance, the induction conditions can be determined as follows.

First, the inductive substance of the promoter is added with varying various addition concentrations and the timing for addition. The cells in which the foreign protein is expressed are harvested, and each of the harvested cells is disrupted, to give a cell free extract.
Each of the resulting extract is subjected to, for instance, SDS-PAGE, and subsequently the bands ascribed to proteins in the gel is visualized by Coomassie brilliant blue staining or silver staining. Among the visualized bands, appropriate induction conditions for the band ascribed to a foreign protein can be examined by determining the concentration of the band by densitometry or other means.

The culture conditions of the cotransformant differs depending upon the cells used as a host, and it is not particularly limited. The level of expressed foreign protein can be determined in the same manner as the determination of the induction conditions by setting various culture time periods and culture temperatures to express the foreign protein under each culture conditions.

The method for isolation and purification of a foreign protein includes, for instance, purification methods for protein as represented by salting-out, ion-exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography and the like.

EXAMPLES
The present invention will be hereinafter described in more detail by means of the following examples, without intending to restrict the scope or spirit of the present invention thereto.

Example 1: Construction of pTf113 A fragment of about 2.6 kb carrying tig gene was cut out with XmnI and NruI from Kohara Clone No. 148 [Kohara, Y. et al., Cell 50, 495-508 (1987)] comprising a trigger factor, and thereafter the resulting XmnI-NruI fragment was blunt-ended, to give a tig gene fragment. pAR3 Plasmid [Perez-Perez, J. & Guitierrez, J., Gene 158, 141-142 (1995)] was cleaved with PstI, and thereafter the resulting linearized plasmid fragment was blunt-ended, to give a pAR3 fragment. The tig gene fragment obtained as above was ligated to the pAR3 fragment, whereby constructing pTf13.

Example 2: Construction of pG-Tfl A fragment of about 2.5 kb carrying tig gene was cut out with Bsp12861I and NruI from Kohara Clone No. 148 mentioned above, and thereafter the resulting Bsp12861I-NruI fragment was blunt-ended, to give a tig gene fragment. pGroll plasmid [Nishihara, K. et al., Rpp1. Environ. Microbiol. 64, 1694-1699 (1988)] was cleaved with SmaI at downstream of groEL gene, and thereafter the resulting blunt-ended tig gene fragment was ligated to the linearized plasmid fragment, to give pG-Tfl.

Preparation Example 1 Preparation of Cotransformant for Expression of Murine Endostatin E. coli BL21 was transformed with pTB01#8 [O'Reilly, M. S. et al., Cell 88, 277-285 (1997); made available from Dr. Thomas Boehm and Dr. Judah Folkman of Children's Hospital, Harvard Medical School] (50 ng) encoding murine endostatin and one of pTf13 or pG-Tfl (each being 50 ng), to give a cotransformant. Here, the transformation was carried out by calcium chloride method.

The cotransformant harboring pTf13 and pTB01#8 was obtained by screening with a plate containing chloramphenicol and ampicillin at concentrations of 20 pg/ml and 50 pg/ml, respectively. The resulting clone in which the trigger factor was coexpressed with the murine endostatin was named NK365.

The cotransformant harboring pG-Tfl and pTB01#8 was obtained by screening with a plate containing chloramphenicol and ampicillin at concentrations of 20 Ng/ml and 50 g/ml, respectively. The resulting clone in which the trigger factor, GroEL and GroES were coexpressed with the murine endostatin was named NK364.

As a comparative example, each of cotransformants, one in which GroEL and GroES were coexpressed with murine endostatin; one in which DnaK, DnaJ and GrpE were coexpressed with murine endostatin; and one in which DnaK, DnaJ, GrpE, GroEL and GroES were coexpressed with murine endostatin, was prepared, respectively.

The clone in which DnaK, DnaJ, GrpE, GroEL and GroES
were coexpressed with murine endostatin was obtained by cotransforming with pG-KJE8 and pTB01#8, and screening with a plate containing chloramphenicol and ampicillin at concentrations of 20 pg/ml and 50 pg/ml, respectively.
The resulting clone was named NK363.

pG-KJE8 was prepared in the manner described below with a plasmid which was capable of regulating chaperone expression more tightly by inserting rrnBTlT2 terminator sequence at downstream of dnaK-dnaJ-grpE gene in pG-KJE6 [Nishihara, K. et al., AppZ. Environ. Microbiol. 64, 1694-1699 (1988)]. First, pKJE7 was cleaved with KpnI at the KpnI site located downstream of dnaK-dnaJ-grpE gene, to give a linearized KpnI-fragment, and thereafter the resulting KpnI-fragment was blunt-ended. Next, rrnBT1T2 sequence cut out from pTrc99A (manufactured by Pharmacia) at the XmnI site was ligated to the blunt-ended fragment, whereby giving plasmid pKJE9. Subsequently, pKJE9 was cleaved at the XmnI site, and blunt-ended tetR-Pztlp-groES-groEL fragment was inserted in the same manner as the case where pG-KJE6 was prepared. A plasmid in which tetR-Pztlp-groES-groEL fragment was inserted in the same orientation as pG-KJE6 was selected, and the plasmid was named pG-KJE8.

Test Example 1: Expression of Murine Endostatin Expression of murine endostatin was examined using each of the cotransformants obtained in Preparation Example 1. The cultivation was carried out using L medium (composition: 1% bactotrypton, 0.5% yeast extract, 0.5%
NaCl, 20 pg/ml chloramphenicol and 50 pg/ml ampicillin).

Each of the cotransformants was cultured at 37 C.
Expression of the chaperone was induced by adding L-arabinose (final concentration: 10 mg/ml) to a medium for NK365 at the beginning of cultivation. Alternatively, expression of the chaperone was induced by adding tetracycline (final concentration: 10 ng/ml) to a medium for NK364. Subsequently, when Klett Unit was about 60, expression of endostatin was induced by adding 10 mM MgSO4 and 3 x 109 pfu/ml X-phage CE6 (manufactured by Novagen) to a cultured medium.

Expression of GroEL and GroES together with murine endostatin was induced by adding tetracycline (50 ng/ml) at the beginning of cultivation for NK363, and adding mM MgSO4 and X-phage CE6 in the same manner as above to a cultured medium when Klett Unit was about 60.

10 In addition, expression of DnaK, DnaJ and GrpE
together with murine endostatin was induced by adding L-arabinose (10 mg/mi) at the beginning of cultivation, and adding each of MgC12 and X-phage CE6 to a cultured medium when Klett Unit was about 60.

Further, expression of DnaK, DnaJ, GrpE, GroEL and GroES together with murine endostatin was induced by adding L-arabinose (10 mg/ml) and tetracycline (20 ng/ml) at the beginning of cultivation, and adding each of MgC12 and X-phage CE6 to a cultured medium when Klett Unit was about 60.

After the induction of expression of endostatin was carried out for 2 hours, the cells were harvested. The resulting cells were subjected to ultrasonic disruption, and thereafter the disrupted cells were centrifuged at 8200 x g, thereby separating a soluble fraction from an insoluble fraction. Each of the fractions was subjected to SDS-PAGE in an amount of 8 pg equivalent of cellular protein. Here, a fraction obtained from NK365, which does not induce expression of the trigger factor, was used as a control. The results are shown in Figure 3.

As shown in the left panel of Figure 3, in the case of coexpressing murine endostatin which is usually insolubilized to be expressed as inclusion bodies in E.
coli, with GroEL and GroES, the majority of endostatin was detected in the soluble fraction, but some murine endostatin was also detected in the insoluble fraction.
Further, also in the case of coexpressing murine endostatin with DnaK, DnaJ and GrpE or coexpressing murine endostatin with DnaK, DnaJ, GrpE, GroEL and GroES, murine endostatin was detected in the insoluble fraction.

On the other hand, from the results in the right panel of Figure 3, in both cases of coexpressing murine endostatin with the trigger factor, and coexpressing murine endostatin with the trigger factor, GroEL and GroES, the expressed endostatin was detected only in the soluble fraction, but not detected in the insoluble fraction. In addition, as compared with the control in which there was no coexpression of a foreign protein together with the trigger factor, or with the trigger factor, GroEL and GroES, it was observed that the soluble fraction was increased.

As described above, as compared with coexpression of each of chaperones GroEL and GroES; DnaK, DnaJ and GrpE;
and DnaK, DnaJ, GrpE, GroEL and GroES, an unexpectedly excellent solubilization effect of a foreign protein can be obtained by coexpression with the trigger factor, or with the trigger factor, GroEL and GroES.

Preparation Example 2 Preparation of Cotransformant for Human ORP150 Expression E. coli JM109 was transformed with plasmid pORP4 (50 ng) encoding human ORP150 and each (50 ng each) of pTf13 or pG-Tfl obtained in Example 1 or 2, and pGrol, which is a plasmid harboring groEL and groES [Nishihara, K. et al., Appl. Environ. Microbiol. 64, 1694-1699 (1988)]. Here, the transformation was carried out by calcium chloride method.

The cotransformant of pTf13 and pORP4 was obtained by screening with a plate containing chloramphenicol and ampicillin at concentrations of 20 g/ml and 50 pg/ml, respectively. The resulting clone was named NK360.

The cotransformant of pG-Tfl and pORP4 was obtained by screening with a plate containing chloramphenicol and ampicillin at concentrations of 20 Ng/ml and 50 pg/ml, respectively. The resulting clone was named NK340.

The cotransformant of pGroll and pORP4 was obtained by screening with a plate containing chloramphenicol and ampicillin at concentrations of 20 pg/ml and 50 pg/ml, respectively. The resulting clone was named NK341.

Test Example 2: Human ORP150 Expression Expression of human ORP150 was examined for each of NK360, NK340 and NK341 obtained in Preparation Example 2.
The cultivation was carried out using L medium (composition: 1% bactotrypton, 0.5% yeast extract, 0.5%
NaCl, 20 pg/ml chloramphenicol and 50 pg/ml ampicillin).
Each of the cotransformants was cultured at 37 C.

Expression of the chaperone and ORP150 was induced by adding tetracycline (final concentration: 10 ng/ml) and IPTG (final concentration: 1 mM) to.each of the cultured media of NK340 and NK341, when Klett Unit reached about 40. Expression of the trigger factor was induced by adding L-arabinose (final concentration: 10 mg/ml)-to a cultured medium of NK360, when Klett Unit reached about 20. Subsequently, expression of ORP150 was induced by adding IPTG (final concentration: 1 mM) thereto, when Klett Unit reached about 40.

Two hours after addition of IPTG, each of the cells was harvested. The resulting cells were subjected to ultrasonic disruption, and thereafter the disrupted cells were centrifuged at 8200 x g, thereby separating a soluble fraction from an insoluble fraction. Each of the fractions was subjected to SDS-PAGE in an amount of 8 pg equivalent of cellular protein. Here, a fraction obtained from NK341, which does not induce expression of the chaperones (GroEL, GroES), was used as a control. The results are shown in Figure 4.

From the results of Figure 4, it is shown that a half amount of expressed ORP150 becomes soluble by coexpressing ORP150, which is usually insolubilized to be expressed as inclusion bodies in E. Coli, with GroEL and GroES or with the trigger factor, and that substantially all of ORP150 become soluble by coexpression with GroEL, GroES and the trigger factor.

The artificial operon and the plasmid of the present invention exhibit excellent properties in that the foreign protein can be expressed in a stabilized state and a solubilized state by coexpression of a desired foreign gene. In addition, the cotransformant of the present invention exhibits an excellent effect in that the foreign protein can be expressed in a stabilized state and a solubilized state. Further, according to a method for producing a foreign protein of the present invention, there can be exhibited an excellent effect that the foreign protein can be expressed in a stabilized state and a solubilized state. According to the present invention, it is made possible to efficiently produce a foreign protein in E. coli by genetic engineering techniques.

SEQUENCE LISTING
GENERAL INFORMATION

APPLICANT: HSP Reseach Institute, Inc.

TITLE OF INVENTION: Trigger Factor Expression Plasmids NUMBER OF SEQUENCES: 7 CORRESPONDENCE ADDRESS: Kirby Eades Gale Baker Box 3432, Station D
Ottawa, ON K1P 6N9 COMPUTER READABLE FORM
COMPUTER: IBM PC Compatible OPERATING SYSTEM: MS-DOS
SOFTWARE:

CURRENT APPLICATION DATA
APPLICATION NUMBER: 2,291,883 FILING DATE: December 24, 1999 CLASSIFICATION:

PRIOR APPLICATION DATA
APPLICATION NUMBER: JPlO-372965 FILING DATE: December 28, 1998 CLASSIFICATION:

PATENT AGENT INFORMATION
NAME: Kimberley Lachaine REFERENCE NUMBER: 44198 INFORMATION FOR SEQ ID NO.: 1 SEQUENCE CHARACTERISTICS
LENGTH: 432 TYPE: PRT

ORIGINAL SOURCE: Escherichia coli SEQUENCE DESCRIPTION: SEQ ID NO.: 1 Met Gln Val Ser Val Glu Thr Thr Gln Gly Leu Gly Arg Arg Val Thr Ile Thr Ile Ala Ala Asp Ser Ile Glu Thr Ala Val Lys Ser Glu Leu Val Asn Val Ala Lys Lys Val Arg Ile Asp Gly Phe Arg Lys Gly Lys Val Pro Met Asn Ile Val Ala Gln Arg Tyr Gly Ala Ser Val Arg Gln Asp Val Leu Gly Asp Leu Met Ser Arg Asn Phe Ile Asp Ala Ile Ile Lys Glu Lys Ile Asn Pro Ala Gly Ala Pro Thr Tyr Val Pro Gly Glu Tyr Lys Leu Gly Glu Asp Phe Thr Tyr Ser Val Glu Phe Glu Val Tyr Pro Glu Val Glu Leu Glu Gly Leu Glu Ala Ile Glu Val Glu Lys Pro Ile Val Glu Val Thr Asp Ala Asp Val Asp Gly Met Leu Asp Thr Leu Arg Lys Gln Gln Ala Thr Trp Lys Glu Lys Asp Gly Ala Val Glu Ala Glu Asp Arg Val Thr Ile Asp Phe Thr Gly Ser Val Asp Gly Glu Glu Phe Glu Gly Gly Lys Ala Ser Asp Phe Val Leu Ala Met Gly Gln Gly Arg Met Ile Pro Gly Phe Glu Asp Gly Ile Lys Gly His Lys Ala Gly Glu Glu Phe Thr Ile Asp Val Thr Phe Pro Glu Glu Tyr His Ala Glu Asn Leu Lys Gly Lys Ala Ala Lys Phe Ala Ile Asn Leu Lys Lys Val Glu Glu Arg Glu Leu Pro Glu Leu Thr Ala Glu Phe Ile Lys Arg Phe Gly Val Glu Asp Gly Ser Val Glu Gly Leu Arg Ala Glu Val Arg Lys Asn Met Glu Arg Glu Leu Arg Ala Pro Ser Val Thr Ala Leu Ser Ser Gln Ala Ile Glu Gly Leu Val Lys Ala Asn Asp Ile Asp Val Pro Ala Ala Leu Ile Asp Ser Glu Ile Asp Val Leu Arg Arg Gln Ala Ala Gln Arg Phe Gly Gly Asn Glu Lys Gln Ala Leu Glu Leu Pro Arg Glu Leu Phe Glu Glu Gln Ala Lys Arg Arg Val Val Val Gly Leu Leu Leu Gly Glu Val Ile Arg Thr Asn Glu Leu Lys Ala Asp Glu Glu Arg Val Lys Gly Leu Ile Glu Glu Met Ala Ser Ala Tyr Glu Asp Pro Lys Glu Val Ile Glu Phe Tyr Ser Lys Asn Lys Glu Leu Met Asp Asn Met Arg Asn Val Ala Leu Glu Glu Gln Ala Val Glu Ala Val Leu Ala Lys Ala Lys Val Thr Glu Lys Glu Thr Thr Phe Asn Glu Leu Met Asn Gln Gln Ala INFORMATION FOR SEQ ID NO.: 2 SEQUENCE CHARACTERISTICS
LENGTH: 1299 TYPE: DNA
ORIGINAL SOURCE: Escherichia coli SEQUENCE DESCRIPTION: SEQ ID NO.: 2 atgcaagttt cagttgaaac cactcaaggc cttggccgcc gtgtaacgat tactatcgct 60 gctgacagca tcgagaccgc tgttaaaagc gagctggtca acgttgcgaa aaaagtacgt 120 attgacggct tccgcaaagg caaagtgcca atgaatatcg ttgctcagcg ttatggcgcg 180 tctgtacgcc aggacgttct gggtgacctg atgagccgta acttcattga cgccatcatt 240 aaagaaaaaa tcaatccggc tggcgcaccg acttatgttc cgggcgaata caagctgggt 300 gaagacttca cttactctgt agagtttgaa gtttatccgg aagttgaact cgagggtctg 360 gaagcgatcg aagttgaaaa accgatcgtt gaagtgaccg acgctgacgt tgacggcatg 420 ctggatactc tgcgtaaaca gcaggcgacc tggaaagaaa aagacggcgc tgttgaagca 480 gaagaccgcg taaccatcga cttcaccggt tctgtagacg gcgaagagtt cgaaggcggt 540 aaagcgtctg atttcgtact ggcgatgggc cagggtcgta tgatcccggg ctttgaagac 600 ggtatcaaag gccacaaagc tggcgaagag ttcaccatcg acgtgacctt cccggaagaa 660 taccacgcag aaaacctgaa aggtaaagca gcgaaattcg ctatcaacct gaagaaagtt 720 gaagagcgtg aactgccgga actgactgca gaattcatca aacgtttcgg cgttgaagat 780 ggttccgtag aaggtctgcg cgctgaagtg cgtaaaaaca tggagcgcga gctgaagagc 840 gccatccgta accgcgttaa gtctcaggcg atcgaaggtc tggtaaaagc taacgacatc 900 gacgtaccgg ctgcgctgat cgacagcgaa atcgacgttc tgcgtcgcca ggctgcacag 960 cgtttcggtg gcaacgaaaa acaagctctg gaactgccgc gcgaactgtt cgaagaacag 1020 gctaaacgcc gcgtagttgt tggcctgctg ctgggcgaag ttatccgcac caacgagctg 1080 aaagctgacg aagagcgcgt gaaaggcctg atcgaagaga tggcttctgc gtacgaagat 1140 ccgaaagaag ttatcgagtt ctacagcaaa aacaaagaac tgatggacaa catgcgcaat 1200 gttgctctgg aagaacaggc tgttgaagct gtactggcga aagcgaaagt gactgaaaaa 1260 gaaaccactt tcaacgagct gatgaaccag caggcgtaa 1299 INFORMATION FOR SEQ ID NO.: 3 SEQUENCE CHARACTERISTICS
LENGTH: 548 TYPE: PRT
ORIGINAL SOURCE: Escherichia coli SEQUENCE DESCRIPTION: SEQ ID NO.: 3 Met Ala Ala Lys Asp Val Lys Phe Gly Asn Asp Ala Arg Val Lys Met Leu Arg Gly Val Asn Val Leu Ala Asp Ala Val Lys Val Thr Leu Gly Pro Lys Gly Arg Asn Val Val Leu Asp Lys Ser Phe Gly Ala Pro Thr Ile Thr Lys Asp Gly Val Ser Val Ala Arg Glu Ile Glu Leu Glu Asp Lys Phe Glu Asn Met Gly Ala Gln Met Val Lys Glu Val Ala Ser Lys Ala Asn Asp Ala Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Gln Ala Ile Ile Thr Glu Gly Leu Lys Ala Val Ala Ala Gly Met Asn Pro Met Asp Leu Lys Arg Gly Ile Asp Lys Ala Val Thr Ala Ala Val Glu Glu Leu Lys Ala Leu Ser Val Pro Cys Ser Asp Ser Lys Ala Ile Ala Gln Val Gly Thr Ile Ser Ala Asn Ser Asp Glu Thr Val Gly Lys Leu Ile Ala Glu Ala Met Asp Lys Val Gly Lys Glu Gly Val Ile Thr Val Glu Asp Gly Thr Gly Leu Gln Asp Glu Leu Asp Val Val Glu Gly Met Gln Phe Asp Arg Gly Tyr Leu Ser Pro Tyr Phe Ile Asn Lys Pro Glu Thr Gly Ala Val Glu Leu Glu Ser Pro Phe Ile Leu Leu Ala Asp Lys Lys Ile Ser Asn Ile Arg Glu Met Leu Pro Val Leu Glu Ala Val Ala Lys Ala Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly Glu Ala Leu Ala Thr Ala Val Val Asn Thr Ile Arg Gly Ile Val Lys Val Ala Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met Leu Gln Asp Ile Ala Thr Leu Thr Gly Gly Thr Val Ile Ser Glu Glu Ile Gly Met Glu Leu Glu Lys Ala Thr Leu Glu Asp Leu Gly Gln Ala Lys Arg Val Val Ile Asn Lys Asp Thr Thr Thr Ile Ile Asp Gly Val Gly Glu Glu Ala Ala Ile Gln Gly Arg Val Ala Gln Ile Arg Gln Gln Ile Glu Glu Ala Thr Ser Asp Tyr Asp Arg Glu Lys Leu Gln Glu Arg Val Ala Lys Leu Ala Gly Gly Val Ala Val Ile Lys Val Gly Ala Ala Thr Glu Val Glu Met Lys Glu Lys Lys Ala Arg Val Glu Asp Ala Leu His Ala Thr Arg Ala Ala Val Glu Glu Gly Val Val Ala Gly Gly Gly Val Ala Leu Ile Arg Val Ala Ser Lys Leu Ala Asp Leu Arg Gly Gln Asn Glu Asp Gln Asn Val Gly Ile Lys Val Ala Leu Arg Ala Met Glu Ala Pro Leu Arg Gln Ile Val Leu Asn Cys Gly Glu Glu Pro Ser Val Val Ala Asn Thr Val Lys Gly Gly Asp Gly Asn Tyr Gly Tyr Asn Ala Ala Thr Glu Glu Tyr Gly Asn Met Ile Asp Met Gly Ile Leu Asp Pro Thr Lys Val Thr Arg Ser Ala Leu Gln Tyr Ala Ala Ser Val Ala Gly Leu Met Ile Thr Thr Glu Cys Met Val Thr Asp Leu Pro Lys Asn Asp Ala Ala Asp Leu Gly Ala Ala Gly Gly Met Gly Gly Met Gly Gly Met Gly Gly Met Met INFORMATION FOR SEQ ID NO.: 4 SEQUENCE CHARACTERISTICS
LENGTH: 97 TYPE: PRT
ORIGINAL SOURCE: Escherichia coli SEQUENCE DESCRIPTION: SEQ ID NO.: 4 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile Val Ile Phe Asn Asp Gly Tyr Gly Val Lys Ser Glu Lys Ile Asp Asn Glu Glu Val Leu Ile Met Ser Glu Ser Asp Ile Leu Ala Ile Val Glu Ala INFORMATION FOR SEQ ID NO.: 5 SEQUENCE CHARACTERISTICS
LENGTH: 1647 TYPE: DNA
ORIGINAL SOURCE: Escherichia coli SEQUENCE DESCRIPTION: SEQ ID NO.: 5 atggcagcta aagacgtaaa attcggtaac gacgctcgtg tgaaaatgct gcgcggcgta 60 aacgtactgg cagatgcagt gaaagttacc ctcggtccaa aaggccgtaa cgtagttctg 120 gataaatctt tcggtgcacc gaccatcacc aaagatggtg tttccgttgc tcgtgaaatc 180 gaactggaag acaagttcga aaatatgggt gcgcagatgg tgaaagaagt tgcctctaaa 240 gcaaacgacg ctgcaggcga cggtaccacc actgcaaccg tactggctca ggctatcatc 300 actgaaggtc tgaaagctgt tgctgcgggc atgaacccga tggacctgaa acgtggtatc 360 gacaaagcgg ttaccgctgc agttgaagaa ctgaaagcgc tgtccgtacc atgctctgac 420 tctaaagcga ttgctcaggt tggtaccatc tccgctaact ccgacgaaac cgtaggtaaa 480 ctgatcgctg aagcgatgga caaagtcggt aaagaaggcg ttatcaccgt tgaagacggt 540 accggtctgc aggacgaact ggacgtggtt gaaggtatgc agttcgaccg tggctacctg 600 tctccttact tcatcaacaa gccggaaact ggcgcagtag aactggaaag cccgttcatc 660 ctgctggctg acaagaaaat ctccaacatc cgcgaaatgc tgccggttct ggaagctgtt 720 gccaaagcag gcaaaccgct gctgatcatc gctgaagatg tagaaggcga agcgctggca 780 actgctgttg ttaacaccat tcgtggcatc gtgaaagtcg ctgcggttaa agcaccgggc 840 ttcggcgatc gtcgtaaagc tatgctgcag gatatcgcaa ccctgactgg cggtaccgtg 900 atctctgaag agatcggtat ggagctggaa aaagcaaccc tggaagacct gggtcaggct 960 aaacgtgttg tgatcaacaa agacaccacc actatcatcg atggcgtggg tgaagaagct 1020 gcaatccagg gccgtgttgc tcagatccgt cagcagattg aagaagcaac ttctgactac 1080 gaccgtgaaa aactgcagga acgcgtagcg aaactggcag gcggcgttgc agttatcaaa 1140 gtgggtgctg ctaccgaagt tgaaatgaaa gagaaaaaag cacgcgttga agatgccctg 1200 cacgcgaccc gtgctgcggt agaagaaggc gtggttgctg gtggtggtgt tgcgctgatc 1260 cgcgtagcgt ctaaactggc tgacctgcgt ggtcagaacg aagaccagaa cgtgggtatc 1320 aaagttgcac tgcgtgcaat ggaagctccg ctgcgtcaga tcgtattgaa ctgcggcgaa 1380 gaaccgtctg ttgttgctaa caccgttaaa ggcggcgacg gcaactacgg ttacaacgca 1440 gcaaccgaag aatacggcaa catgatcgac atgggtatcc tggatccaac caaagtaact 1500 cgttctgctc tgcagtacgc agcttctgtg gctggcctga tgatcaccac cgaatgcatg 1560 gttaccgacc tgccgaaaaa cgatgcagct gacttaggcg ctgctggcgg tatgggcggc 1620 atgggtggca tgggcggcat qatgtaa 1647 INFORMATION FOR SEQ ID NO.: 6 SEQUENCE CHARACTERISTICS
LENGTH: 294 TYPE: DNA
ORIGINAL SOURCE: Escherichia coli SEQUENCE DESCRIPTION: SEQ ID NO.: 6 atgaatattc gtccattgca tgatcgcgtg atcgtcaagc gtaaagaagt tgaaactaaa 60 tctgctggcg gcatcgttct gaccggc,tct gcagcggcta aatccacccg cggcgaagtg 120 ctggctgtcg gcaatggccg tatccttgaa aatggcgaag tgaagccgct ggatgtgaaa 180 gttggcgaca tcgttatttt caacgatggc tacggtgtga aatctgagaa gatcgacaat 240 gaagaagtgt tgatcatgtc cgaaagcgac attctggcaa ttgttgaagc gtaa 294 INFORMATION FOR SEQ ID NO.: 7 SEQUENCE CHARACTERISTICS
LENGTH: 4525 TYPE: DNA
ORIGINAL SOURCE: Escherichia coli SEQUENCE DESCRIPTION: SEQ ID NO.: 7 ggcgtcaccc ataacagata cggactttct caaaggagag ttatcaatga atattcgtcc 60 attgcatgat cgcgtgatcg 'tcaagcgtaa agaagttga.a actaaatctg ctggcggcat 120 cgttctgacc ggctctgcag cggctaaatc cacccgcggc gaagtgctgg ctgtcggcaa 180 tggccgtatc cttgaaaatg gcgaagtgaa gccgctgga.t gtgaaagttg gcgacatcgt 240 tattttcaac gatggctacg gtgtgaaatc tgagaagatc gacaatgaag aagtgttgat 300 catgtccgaa agcgacattc tggcaattgt tgaagcgtaa tccgcgcacg acactgaaca 360 tacgaattta aggaataaag ataatggcag ctaaagacgt aaaattcggt aacgacgctc 420 gtgtgaaaat gctgcgcggc gtaaacgtac tggcagatgc agtgaaagtt accctcggtc 480 caaaaggccg taacgtagtt ctggataaat ctttcggtgc accgaccatc accaaagatg 540 gtgtttccgt tgctcgtgaa atcgaactgg aagacaagtt cgaaaatatg ggtgcgcaga 600 tggtgaaaga agttgcctct aaagcaaacg acgctgcagg cgacggtacc accactgcaa 660 ccgtactggc tcaggctatc atcactgaag gtctgaaagc tgttgctgcg ggcatgaacc 720 cgatggacct gaaacgtggt atcgacaaag cggttaccgc tgcagttgaa gaactgaaag 780 cgctgtccgt accatgctct gactctaaag cgattgctca ggttggtacc atctccgcta 840 actccgacga aaccgtaggt aaactgatcg ctgaagcgat ggacaaagtc ggtaaagaag 900 gcgttatcac cgttgaagac ggtaccggtc tgcaggacga actggacgtg gttgaaggta 960 tgcagttcga ccgtggctac ctgtctcctt acttcatcaa caagccggaa actggcgcag 1020 tagaactgga aagcccgttc atcctgctgg ctgacaagaa aatctccaac atccgcgaaa 1080 tgctgccggt tctggaagct gttgccaaag caggcaaacc gctgctgatc atcgctgaag 1140 atgtagaagg cgaagcgctg gcaactgctg ttgttaacac cattcgtggc atcgtgaaag 1200 tcgctgcggt taaagcaccg ggcttcggcg atcgtcgtaa agctatgctg caggatatcg 1260 caaccctgac tggcggtacc gtgatctctg aagagatcgg tatggagctg gaaaaagcaa 1320 ccctggaaga cctgggtcag gctaaacgtg ttgtgatcaa caaagacacc accactatca 1380 tcgatggcgt gggtgaagaa gctgcaatcc agggccgtgt tgctcagatc cgtcagcaga 1440 ttgaagaagc aacttctgac tacgaccgtg aaaaactgca ggaacgcgta gcgaaactgg 1500 caggcggcgt tgcagttatc aaagtgggtg ctgctaccga agttgaaatg aaagagaaaa 1560 aagcacgcgt tgaagatgcc ctgcacgcga cccgtgctgc ggtagaagaa ggcgtggttg 1620 ctggtggtgg tgttgcgctg atccgcgtag cgtctaaact ggctgacctg cgtggtcaga 1680 acgaagacca gaacgtgggt aGcaaagttg cactgcgtgc aatggaagct ccgctgcgtc 1740 agatcgtatt gaactgcggc gaagaaccgt ctgttgttgc taacaccgtt aaaggcggcg 1800 acggcaacta cggttacaac gcagcaaccg aagaatacgg caacatgatc gacatgggta 1860 tcctggatcc aaccaaagta actcgttctg ctctgcagta cgcagcttct gtggctggcc 1920 tgatgatcac caccgaatgc atggttaccg acctgccgaa aaacgatgca gctgacttag 1980 gcgctgctgg cggtatgggc ggcatgggtg gcatgggcgg catgatgtaa ttgccctgca 2040 cctcgcagaa ataaacaaac ccccctgtga ttttttgagg taacaagatg caagtttcag 2100 ttgaaaccac tcaaggcctt ggccgccgtg taacgattac tatcgctgct gacagcatcg 2160 agaccgctgt taaaagcgag ctggtcaacg ttgcgaaaaa agtacgtatt gacggcttcc 2220 gcaaaggcaa agtgccaatg aatatcgttg ctcagcgtta tggcgcgtct gtacgccagg 2280 acgttctggg tgacctgatg agccgtaact tcattgacgc catcattaaa gaaaaaatca 2340 atccggctgg cgcaccgact tatgttccgg gcgaatacaa gctgggtgaa gacttcactt 2400 actctgtaga gtttgaagtt tatccggaag ttgaactcga gggtctggaa gcgatcgaag 2460 ttgaaaaacc gatcgttgaa gtgaccgacg ctgacgttga cggcatgctg gatactctgc 2520 gtaaacagca ggcgacctgg aaagaaaaag acggcgctgt tgaagcagaa gaccgcgtaa 2580 ccatcgactt caccggttct gtagacggcg aagagttcga aggcggtaaa gcgtctgatt 2640 tcgtactggc gatgggccag ggtcgtatga tcccgggctt tgaagacggt atcaaaggcc 2700 acaaagctgg cgaagagttc accatcgacg tgaccttccc ggaagaatac cacgcagaaa 2760 acctgaaagg taaagcagcg aaattcgcta tcaacctgaa gaaagttgaa gagcgtgaac 2820 tgccggaact gactgcagaa ttcatcaaac gtttcggcgt tgaagatggt tccgtagaag 2880 gtctgcgcgc tgaagtgcgt aaaaacatgg agcgcgagct gaagagcgcc atccgtaacc 2940 gcgttaagtc tcaggcgatc gaaggtctgg taaaagctaa cgacatcgac gtaccggctg 3000 cgctgatcga cagcgaaatc gacgttctgc gtcgccaggc tgcacagcgt ttcggtggca 3060 acgaaaaaca agctctggaa ctgccgcgcg aactgttcga agaacaggct aaacgccgcg 3120 tagttgttgg cctgctgctg ggcgaagtta tccgcaccaa cgagctgaaa gctgacgaag 3180 agcgcgtgaa aggcctgatc gaagagatgg cttctgcgta cgaagatccg aaagaagtta 3240 tcgagttcta cagcaaaaac aaagaactga tggacaacat gcgcaatgtt gctctggaag 3300 aacaggctgt tgaagctgta ctggcgaaag cgaaagtgac tgaaaaagaa accactttca 3360 acgagctgat gaaccagcag gcgtaattta cgcagcataa cgcgctaaat tcgcacaaag 3420 gcccgtcacc gccaggtggt gggctttttt ttgtcatgaa ttttgcatgg aaccgtgcga 3480 aaagcctctt tcggtgttag cgtaacaaca aaagattgtt atgcttgaaa tatggtgatg 3540 ccgtacccat aacacaggga ctagctgata atccgtccat aaggttacaa tcggtacagc 3600 aggttttttc aattttatcc aggagacgga aatgtcatac agcggcgaac gagataactt 3660 tgcaccccat atggcgctgg tgccgatggt cattgaacag acctcacgcg gtgagcgctc 3720 ttttgatatc tattctcgtc tacttaagga acgcgtcatt tttctgactg gccaggttga 3780 agaccacatg gctaacctga ttgtggcgca gatgctgttc ctggaagcag aaaacccaga 3840 aaaagatatc tatctgtaca ttaactcccc aggcggggtg atcactgccg ggatgtctat 3900 ctatgacacc atgcagttta tcaagcctga tgtcagcacc atctgtatgg gccaggcggc 3960 ctcgatgggc gctttcttgc tgaccgcagg ggcaaaaggt aaacgttttt gcctgccgaa 4020 ttcgcgcgtg atgattcacc aaccgttggg cggctaccag ggccaggcga ccgatatcga 4080 aattcatgcc cgtgaaattc tgaaagttaa agggcgcatg aatgaactta tggcgcttca 4140 tacgggtcaa tcattagaac agattgaacg tgataccgag cgcgatcgct tcctttccgc 4200 ccctgaagcg gtggaatacg gtctggtcga ttcgattctg acccatcgta attgatgcca 4260 gaggcgcaac tgtgccgcta tacttatcca gggcggcaca acgctgtaag cgcttgcgcc 4320 tgagaatggc atttgcgtcg tcgtgtgcgg cacaaagaac aaagaagagg ttttgaccca 4380 tgacagataa acgcaaagat ggctcaggca aattgctgta ttgctctttt tgcggcaaaa 4440 gccagcatga agtgcgcaag ctgattgccg gtccatccgt gtatatctgc gacgaatgtg 4500 ttgatttatg taacgacatc attcg 4525

Claims (13)

1. An artificial operon comprising a gene encoding a trigger factor, a gene encoding GroEL, and a gene encoding GroES.

2. The artificial operon according to claim 1, further comprising an inducible promoter.

3. The artificial operon according to claim 2, wherein said inducible promoter is selected from the group consisting of lac, trp, ara and Pzt-1.

4. A plasmid expressing each of a trigger factor, GroEL and GroES, the plasmid carrying the artificial operon according to any one of claims 1 to 3.

5. An isolated cotransformed cell harboring the plasmid according to claim 4 and an expression plasmid for a foreign protein.

6. The cotransformed cell according to claim 5, obtained by using a protease mutant of E. coli as a host.

7. The cotransformed cell according to claim 6, wherein the protease mutant is a lon-clpPX double mutant or a lon-clpPX-hslV/U triple mutant.

8. The cotransformed cell according to claim 5, obtained by using a plsX mutant of E. coli as a host.

9. The cotransformed cell according to claim 5, obtained by using an rpoH mutant of E. coli as a host.

10. The cotransformed cell according to claim 9, wherein said rpoH mutant is an rpoH deletion mutant.

11. The cotransformed cell according to any one of claims 5 to 10, wherein said foreign protein is selected from the group consisting of interferons, interleukins, interleukin receptors, interleukin receptor antagonists, granulocyte colony-stimulating factors, granulocyte macrophage colony-stimulating factors, macrophage colony-stimulating factors, erythropoietin, thrombopoietin, leukemia inhibitory factors, stem cell growth factors, tumor necrosis factors, growth hormones, proinsulin, insulin-like growth factors, fibroblast growth factors, platelet-derived growth factors, transforming growth factors, hepatocyte growth factors, osteogenic factors, nerve growth factors, ciliary neurotrophic factors, brain-derived neurotrophic factors, glial-derived neurotrophic factors, neurotrophine, angiogenesis inhibitors, prourokinase, tissue plasminogen activators, blood coagulation factors, protein C, glucocerebrosidase, superoxide dismutase, renin, lysozyme, P450, prochymosin, trypsin inhibitors, elastase inhibitors, lipocortin, reptin, immunoglobulins, single-chain antibodies, complement components, serum albumin, cedar pollen allergens, hypoxia-induced stress proteins, protein kinases, proto-oncogene products, transcription regulation factors and virus-constituent proteins.

12. A method for producing a foreign protein comprising the step of:

culturing the cotransformed cell according to any one of claims 5 to 11.

13. The method according to claim 12, wherein the cotransformed cell is cultured under the induction conditions that an expression level of the trigger factor, or an expression level of each of the trigger factor, GroEL and GroES are at a level for solubilization of the foreign protein.